Treatment of inflammatory bowel diseases with mammal beta defensins

ABSTRACT

The present invention relates to treatment of inflammatory bowel diseases with mammal beta defensins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 12/504,909filed Jul. 17, 2009, which claims priority or the benefit under 35U.S.C. 119 of European application nos. 08160761.6, 08162486.8 and08163614.4 filed Jul. 18, 2008, Aug. 15, 2008, and Sep. 3, 2008,respectively, and U.S. provisional application Nos. 61/086,910,61/090,937 and 61/094,556 filed Aug. 7, 2008, Aug. 22, 2008, and Sep. 5,2008, respectively, the contents of which are fully incorporated hereinby reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form,which is incorporated herein by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to prevention and treatment ofinflammatory bowel diseases by administration of a mammal beta defensin.

2. Background

Human Defensins

Among many other elements, key components of innate immunity are theantimicrobial peptides (AMPs) that individually show considerableselectivity, but collectively are able to rapidly kill a broad spectrumof bacteria, viruses and fungi. The biological significance of AMPs isemphasized by their ubiquitous distribution in nature and they areprobably produced by ail multicellular organisms. In humans thepredominant AMPs are the defensins. The human defensins are smallcationic peptides that can be divided into α- and β-defensins based onthe topology of their three intramolecular cysteine disulphide bonds.The α-defensins can be further subdivided into those that were firstisolated from neutrophil granules (HNP1-4) and those that are expressedby Paneth cells in the crypts of the small intestine (HD5 and HD6). Theβ-defensins are mainly produced by epithelial cells in various oftissues and organs including the skin, trachea, gastrointestinal tract,urogenital system, kidneys, pancreas and mammary gland. The bestcharacterized members of the β-defensin family are hBD1-3. However,using various bioinformatics tools almost 40 open reading framesencoding putative β-defensin homologues have been annotated in the humangenome. Some of the human defensins are produced constitutively, whereasothers are induced by proinflammatory cytokines or exogenous microbialproducts.

It has become increasingly clear that the human defensins in addition totheft direct antimicrobial activity also have a wide range ofimmunomodulatory/alternative properties. These include the induction ofvarious chemokines and cytokines, chemotactic and apoptotic activities,induction of prostaglandin, histamine and leukotriene release,inhibition of complement, stimulation of dendritic cell maturationthrough toll-like receptor signaling and stimulation of pathogenclearance by neutrophils. Furthermore, the human defensins also play arole in wound healing, proliferation of epithelial and fibroblast cells,angiogenesis and vasculogenesis.

There is increasing evidence that the human defensins play an importantrole in many infectious and inflammatory diseases. Overexpression ofhuman defensins is often observed in inflamed and/or infected skin mostlikely because of local induction by microbial components or endogenousproinflammatory cytokines. In psoriasis hBD2 and hBD3 are overabundantand in lesional epithelium of patients with acne vulgaris or superficialfolliculitis a significant upregulation of hBD2 has been observed. Onthe other hand, downregulation of hBD2 and hBD3 has been associated withatopic dermatitis. Heal Crohn's disease has been associated withdeficient expression of HD5 and HD6 and in Crohn's disease in the colonexpression of hBD2-4 are down-regulated.

Cytokines

Cytokines are small, secreted polypeptides from higher eukaryotes whichare responsible for intercellular signal transduction and which affectthe growth, division and functions of other cells. They are potent,pleiotropic polypeptides that, e.g. via corresponding receptors, act aslocal or systemic intercellular regulatory factors, and therefore playcrucial roles in many biologic processes, such as immunity,inflammation, and hematopoiesis. Cytokines are produced by diverse celltypes including fibroblasts, endothelial cells, epithelial cells,macrophages/monocytes, and lymphocytes.

TNF-α is implicated in various pathophysiological processes and can beeither protective, as in host defense, or deleterious, as inautoimmunity. TNF-α is one of the key cytokines that triggers andsustains the inflammation response and TNF-α inactivation has proven tobe important in downregulating the inflammatory reactions associatedwith autoimmune diseases. Upon an infection, TNF-α is secreted in highamounts by macrophages and it mediates the recruitment of neutrophilsand macrophages to sites of infection by stimulating endothelial cellsto produce adhesion molecules and by producing chemokines, which arechemotactic cytokines. TNF-α help activate leukocytes and otherinflammatory cells and increase vascular permeability within injuredtissues. TNF-α is mainly produced by macrophages, monocytes anddendritic cells, but also by a broad variety of other cell typesincluding lymphoid cells, mast cells, endothelial cells, cardiacmyocytes, adipose tissue, fibroblasts and neuronal tissue.

Current anti-inflammatory drugs block the action of TNF-α by binding toit and hereby prevents it from signaling the receptors for TNF-α on thesurface of cells. This type of blocking has some serious side effects,of which some is infections such as tuberculosis, sepsis and fungalinfections and possible increased cancer incidence.

IL-10, also known as human cytokine synthesis inhibitory factor (CSIF),is also a key player in immune regulation as an anti-inflammatorycytokine. This cytokine is produced by several cell types includingmonocytes, macrophages, T cells, B cells, dendritic cells and mastcells. This cytokine has pleiotropic effects in immunoregulation andinflammation. It down-regulates the expression of pro-inflammatorycytokines, cytokines secreted by Th1/Th17 cells, MHC class II Ags, andcostimulatory molecules on antigen-presenting cells. IL-10 is alsosecreted by a population of T cells called regulatory T cells (Tregs).These cells do not prevent initial T cell activation; rather, theyinhibit a sustained response and prevent chronic and potentiallydamaging responses. In the periphery some T cells are induced to becomeTregs by antigen and either IL-10 or TGF-β. Tregs induced by IL-10 areCD4+/CD25+/Foxp3− and are referred to as Tr1 cells. These cells suppressimmune responses by secretion of IL-10.

Recent studies have revealed a greater diversification of the T celleffector repertoire than the Th1/Th2/Treg with the identification ofTh17 cells. This subpopulation has been shown to be pathogenic inseveral autoimmune diseases, such as Crohn's disease, ulcerativecolitis, psoriasis and multiple sclerosis, previously attributed to theTh1 lineage. The cytokines secreted by Th17 are also downregulated byIL-10 and blocking of TNF prevents psoriasis by inactivating Th17 cells.The overall activity of IL-10 is anti-inflammatory and it has been shownto prevent inflammation and injury in several animal studies, howeverclinical IL-10 treatment remains insufficient because of difficulties inthe route of IL-10 administration and its biological half-life.

Inflammatory Bowel Diseases

Inflammatory bowel diseases (IBD) are defined by chronic, relapsingintestinal inflammation of obscure origin. IBD refers to two distinctdisorders, Crohn's disease and ulcerative colitis (UC). Both diseasesappear to result from the unrestrained activation of an inflammatoryresponse in the intestine. This inflammatory cascade is thought to beperpetuated through the actions of proinflammatory cytokines andselective activation of lymphocyte subsets. In patients with IBD, ulcersand inflammation of the inner lining of the intestines lead to symptomsof abdominal pain, diarrhea, and rectal bleeding. Ulcerative colitisoccurs in the large intestine, while in Crohn's, the disease can involvethe entire GI tract as well as the small and large intestines. For mostpatients. IBD is a chronic condition with symptoms lasting for months toyears. It is most common in young adults, but can occur at any age. Itis found worldwide, but is most common in industrialized countries suchas the United States, England, and northern Europe. It is especiallycommon in people of Jewish descent and has racial differences inincidence as well. The clinical symptoms of IBD are intermittent rectalbleeding, crampy abdominal pain, weight loss and diarrhea. Diagnosis ofIBD is based on the clinical symptoms, the use of a barium enema, butdirect visualization (sigmoidoscopy or colonoscopy) is the most accuratetest. Protracted IBD is a risk factor for colon cancer, and treatment ofIBD can involve medications and surgery.

Some patients with UC only have disease in the rectum (proctitis).Others with UC have disease limited to the rectum and the adjacent leftcolon (proctosigmoiditis). Yet others have UC of the entire colon(universal IBD). Symptoms of UC are generally more severe with moreextensive disease (larger portion of the colon involved with disease).

The prognosis for patients with disease limited to the rectum(proctitis) or UC limited to the end of the left colon(proctosigmoiditis) is better then that of full colon UC. Brief periodictreatments using oral medications or enemas may be sufficient. In thosewith more extensive disease, blood loss from the inflamed intestines canlead to anemia, and may require treatment with iron supplements or evenblood transfusions. Rarely, the colon can acutely dilate to a large sizewhen the inflammation becomes very severe. This condition is calledtoxic megacolon. Patients with toxic megacolon are extremely ill withfever, abdominal pain and distention, dehydration, and malnutrition.Unless the patient improves rapidly with medication, surgery is usuallynecessary to prevent colon rupture.

Crohn's disease can occur in all regions of the gastrointestinal tract.With this disease intestinal obstruction due to inflammation andfibrosis occurs in a large number of patients. Granulomas and fistulaformation are frequent complications of Crohn's disease. Diseaseprogression consequences include intravenous feeding, surgery andcolostomy.

IBD may be treated medicinally. The most commonly used medications totreat IBD are anti-inflammatory drugs such as the salicylates. Thesalicylate preparations have been effective in treating mild to moderatedisease. They can also decrease the frequency of disease flares when themedications are taken on a prolonged basis. Examples of salicylatesinclude sulfasalazine, olsalazine, and mesalamine. All of thesemedications are given orally in high doses for maximal therapeuticbenefit. These medicines are not without side effects. Azulfidine cancause upset stomach when taken in high doses, and rare cases of mildkidney inflammation have been reported with some salicylatepreparations.

Corticosteroids are more potent and faster-acting than salicylates inthe treatment of IBD, but potentially serious side effects limit the useof corticosteroids to patients with more severe disease. Side effects ofcorticosteroids usually occur with long term use. They include thinningof the bone and skin, infections, diabetes, muscle wasting, rounding offaces, psychiatric disturbances, and, on rare occasions, destruction ofhip joints.

In IBD patients that do not respond to salicylates or corticosteroids,medications that suppress the immune system are used. Examples ofimmunosuppressants include azathioprine and 6-mercaptopurine.Immunosuppressants used in this situation help to control IBD and allowgradual reduction or elimination of corticosteroids. However,immunosuppressants render the patient immuno-compromised and susceptibleto many other diseases.

A well recognized model for studying IBD is the DSS colitis mouse model,as described in Kawada et al. “Insights from advances in research ofchemically induced experimental models of human inflammatory boweldisease”, World J. Gastroenterol. 13(42): 5581-5593 (2007); and Wirtzand Neurath “Mouse models of inflammatory bowel disease”, Advanced DrugDelivery Reviews 59(11): 1073-1083 (2007).

Clearly there is a great need for agents capable of preventing andtreating IBD.

Using Human Defensins to Treat Inflammatory Bowel Diseases

Interestingly, Crohn's disease in the small intestine has beenassociated with decreased levels of the paneth cell α-defensins HD5 andHD6, whereas Crohn's disease in the colon has been associated withreduced production of the β-defensins hBD2 and hBD3 (Gersemann at al.,2008; Wehkamp et al., 2005). Furthermore, involvement of the entericmicrobiota in the pathogenesis of Crohn's has been convincinglydemonstrated (Swidsinski at al., 2002). Using fluorescence in situhybridization, these researchers showed that in active Crohn's disease adrastic increase of mucosa-associated and invasive bacteria wasobserved, whereas these bacteria are absent from the normal small andlarge bowel epithelium. Together these observations have merged into ahypothesis, which suggest that in healthy persons a proper level ofdefensins along the intestinal epithelial barrier acts to control thecomposition and number of luminal bacteria and keep them away fromadhering to and invading the mucosa to trigger an inflammation (Wang etal., 2007). On the other hand, in persons with an insufficient abilityto produce a protective level of secreted defensins, the balance isshifted between the antimicrobial defence and the luminal bacteria. As aresult, this allows a bacterial invasion into underlying intestinaltissues that induce an inflammatory state, which in turn, may developinto Crohn's disease.

Based on this hypothesis, WO 2007/081486 discloses the use of severalhuman defensins in the treatment of inflammatory bowel disease. Theinventors suggested that defensins administered orally to Crohn'spatients, in a formulation that allow their release at proper locationsin the intestinal lumen, would reduce the number of invading bacteria,re-establish a normal epithelial barrier function and, thus, reduce theseverity of the inflammatory disease.

According to WO 2007/081486, the function of the defensins is todirectly target and kill bacteria in the lumen to prevent them frominvading the epithelial tissue. That is, the function of the defensinsis purely as an anti-infective compound. In relation to WO 2007/081486,it is surprising that hBD2 administered parentally is able to reduce theseverity of DSS induced colitis in mice, because by using this route ofadministration the peptide never encounters luminal bacteria.Additionally, we show here that the effect of hBD2 is a reduction of thelevel of the pro-inflammatory cytokines TNFα, IL-1β and IL-23 secretedby PBMCs. These cytokines are known to be key players in manyinflammatory diseases including inflammatory bowel disease. It has beenknown for more than a decade that the defensins beside theiranti-microbial functions also posses a range of immunomodulatoryfunctions. However, the large majority of work on the immune modulatingproperties of the human defensins describes them as having primarilypro-inflammatory or immune enhancing functions (See for example,Niyonsaba et al., 2007; Bowdish at al., 2006; Lehrer, 2004).

Hence, it is truly unexpected that hBD2 administered parentally shouldbe able to reduce disease severity in IBD patients. First of all, whenadministered parentally, hBD2 would never reach the intestinal lumen toencounter harmful bacteria involved in inducing the disease. Moreover,based on the large majority of published literature, one would expectthat a defensin entering the blood stream would induce apro-inflammatory rather than an anti-inflammatory response, as observedin the work presented here.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Defensin: The term “defensin” as used herein refers to polypeptidesrecognized by a person skilled in the art as belonging to the defensinclass of antimicrobial peptides. To determine if a polypeptide is adefensin according to the invention, the amino acid sequence may becompared with the hidden markov model profiles (HMM profiles) of thePFAM database by using the freely available HMMER software package.

The PFAM defensin families include for example Defensin_(—)1 or“Mammalian defensin” (accession no. PF00323), and Defensin_(—)2 orDefensin_beta or “Beta Defensin” (accession no. PF00711).

The defensins of the invention belong to the beta defensin class. Thedefensins from the beta defensin class share common structural features,such as the cysteine pattern.

Examples of defensins, according to the invention, include human betadefensin 1 (hBD1; see SEQ ID NO:1), human beta defensin 2 (hBD2; see SEQID NO:2), human beta defensin 3 (hBD3; see SEQ ID NO:3), human betadefensin 4 (hBD4; see SEQ ID NO:4), and mouse beta defensin 3 (mBD3; seeSEQ ID NO:6).

Identity: The relatedness between two amino acid sequences or betweentwo nucleotide sequences is described by the parameter “identity”.

For purposes of the present invention, the degree of identity betweentwo amino acid sequences is determined using the Needleman-Wunschalgorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice et al., 2000,Trends in Genetics 16: 276-277; http://emboss.org), preferably version3.0.0 or later. The optional parameters used are gap open penalty of 10,gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version ofBLOSUM62) substitution matrix. The output of Needle labeled “longestidentity” (obtained using the -nobrief option) is used as the percentidentity and is calculated as follows:

(Identical Residues×100)/(Length of Alignment−Total Number of Gaps inAlignment)

For purposes of the present invention, the degree of identity betweentwo deoxyribonucleotide sequences is determined using theNeedleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) asimplemented in the Needle program of the EMBOSS package (EMBOSS: TheEuropean Molecular Biology Open Software Suite, Rice at al., 2000,supra; http://emboss.org), preferably version 3.0.0 or later. Theoptional parameters used are gap open penalty of 10, gap extensionpenalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4)substitution matrix. The output of Needle labeled “longest identity”(obtained using the -nobrief option) is used as the percent identity andis calculated as follows:

(Identical Deoxyribonucleotides×100)/(Length of Alignment−Total Numberof Gaps in Alignment).

Isolated polypeptide: The term “isolated variant” or “isolatedpolypeptide” as used herein refers to a variant or a polypeptide that isisolated from a source. In one aspect, the variant or polypeptide is atleast 1% pure, preferably at least 5% pure, more preferably at least 10%pure, more preferably at least 20% pure, more preferably at least 40%pure, more preferably at least 60% pure, even more preferably at least80% pure, and most preferably at least 90% pure, as determined bySDS-PAGE.

Substantially pure polypeptide: The term “substantially purepolypeptide” denotes herein a polypeptide preparation that contains atmost 10%, preferably at most 8%, more preferably at most 6%, morepreferably at most 5%, more preferably at most 4%, more preferably atmost 3%, even more preferably at most 2%, most preferably at most 1%,and even most preferably at most 0.5% by weight of other polypeptidematerial with which it is natively or recombinantly associated. It is,therefore, preferred that the substantially pure polypeptide is at least92% pure, preferably at least 94% pure, more preferably at least 95%pure, more preferably at least 96% pure, more preferably at least 96%pure, more preferably at least 97% pure, more preferably at least 98%pure, even more preferably at least 99%, most preferably at least 99.5%pure, and even most preferably 100% pure by weight of the totalpolypeptide material present in the preparation. The polypeptides of thepresent invention are preferably in a substantially pure form. This canbe accomplished, for example, by preparing the polypeptide by well-knownrecombinant methods or by classical purification methods.

Mammal Beta Defensins

The present invention relates to pharmaceutical uses of mammal betadefensins, such as human beta defensins and/or mouse beta defensins, inthe treatment of inflammatory bowel diseases, such as ulcerative colitisand/or Crohns disease. The treatment is preferably associated withreduced TNF-alpha activity in treated tissues.

In an embodiment, the mammal beta defensins of the invention have adegree of identity of at least 80%, preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% to any of theamino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5 and/or SEQ ID NO:6. In a preferred embodiment, themammal beta defensins of the invention have a degree of identity of atleast 80%, preferably at least 85%, more preferably at least 90%, andmost preferably at least 95% to any of the amino acid sequences of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3 and/or SEQ ID NO:4. In a morepreferred embodiment, the mammal beta defensins of the invention consistof human beta defensin 1 (SEQ ID NO:1), human beta defensin 2 (SEQ IDNO:2), human beta defensin 3 (SEQ ID NO:3), human beta defensin 4 (SEQID NO:4), a variant of human beta defensin 4 (SEQ ID NO:5) and/or mousebeta defensin 3 (SEQ ID NO:6). In an even more preferred embodiment, themammal beta defensins of the invention consist of human beta defensin 1(SEQ ID NO:1), human beta defensin 2 (SEQ ID NO:2), human beta defensin3 (SEQ ID NO:3) and/or human beta defensin 4 (SEQ ID NO:4).

In another embodiment, the mammal beta defensins of the invention have adegree of identity of at least 80%, preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% to the aminoacid sequence of SEQ ID NO:2. In a preferred embodiment, the mammal betadefensins of the invention consist of human beta defensin 2 (SEQ IDNO:2).

In yet another embodiment, the mammal beta defensins of the inventionconsist of human beta defensins and/or mouse beta defensins, andfunctionally equivalent variants thereof. Preferably, the mammal betadefensins consist of human beta defensin 1, human beta defensin 2, humanbeta defensin 3, human beta defensin 4 and mouse beta defensin 3, andfunctionally equivalent variants thereof. More preferably, the mammalbeta defensins of the invention consist of human beta defensin 2, andfunctionally equivalent variants thereof.

The mammal beta defensins of the invention are also referred to ascompounds of the preferred embodiments.

In the context of the present invention, a “functionally equivalentvariant” of a mammal (e.g. human) beta defensin is a modified mammal(e.g. human) beta defensin exhibiting approx. the same effect on aninflammatory bowel disease as the parent mammal (e.g. human) betadefensin. Preferably, it also exhibits approx. the same effect onTNF-alpha activity as the mammal (e.g. human) beta defensin.

According to the invention, a functionally equivalent variant of amammal (e.g. human) beta defensin may comprise 1-5 amino acidmodifications, preferably 1-4 amino acid modifications, more preferably1-3 amino acid modifications, most preferably 1-2 amino acidmodification(s), and in particular one amino acid modification, ascompared to the mammal (e.g. human) beta defensin amino acid sequence.

The term “modification” means herein any chemical modification of amammal (e.g. human) beta defensin. The modification(s) can besubstitution(s), deletion(s) and/or insertions(s) of the amino acid(s)as well as replacement(s) of amino acid side chain(s); or use ofunnatural amino acids with similar characteristics in the amino acidsequence. In particular the modification(s) can be amidations, such asamidation of the C-terminus.

Preferably, amino add modifications are of a minor nature, that isconservative amino acid substitutions or insertions that do notsignificantly affect the folding and/or activity of the polypeptide;single deletions; small amino- or carboxyl-terminal extensions; a smalllinker peptide of up to about 20-25 residues; or a small extension thatfacilitates purification by changing net charge or another function,such as a poly-histidine tag, an antigenic epitope or a binding domain.

Examples of conservative substitutions are within the group of basicamino acids (arginine, lysine and histidine), acidic amino acids(glutamic acid and aspartic acid), polar amino acids (glutamine andasparagine), hydrophobic amino acids (leucine, isoleucine and valine),aromatic amino acids (phenylalanine, tryptophan and tyrosine), and smallamino acids (glycine, alanine, serine, threonine and methionine). Aminoacid substitutions which do not generally alter specific activity areknown in the art and are described, for example, by H. Neurath and R. L.Hill, 1979, In, The Proteins, Academic Press, New York. The mostcommonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser,Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg,Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, and Asp/Gly.

In addition to the 20 standard amino acids, non-standard amino acids(such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid,isovaline, and alpha-methyl serine) may be substituted for amino acidresidues of a wild-type polypeptide. A limited number ofnon-conservative amino acids, amino acids that are not encoded by thegenetic code, and unnatural amino acids may be substituted for aminoacid residues. “Unnatural amino acids” have been modified after proteinsynthesis, and/or have a chemical structure in their side chain(s)different from that of the standard amino acids. Unnatural amino acidscan be chemically synthesized, and preferably, are commerciallyavailable, and include pipecolic acid, thiazolidine carboxylic acid,dehydroproline, 3- and 4-methylproline, and 3,3-dimethylproline.

Essential amino acids in a mammal beta defensin can be identifiedaccording to procedures known in the art, such as site-directedmutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989,Science 244: 1081-1085). In the latter technique, single alaninemutations are introduced at every residue in the molecule, and theresultant mutant molecules are tested for biological activity (i.e.,activity against an inflammatory bowel disease and/or suppression ofTNF-alpha activity) to identify amino acid residues that are critical tothe activity of the molecule. See also, Hilton et al., 1996, J. Biol.Chem. 271: 4699-4708. The identities of essential amino acids can alsobe inferred from analysis of identities with polypeptides which arerelated to mammal beta defensins.

Single or multiple amino acid substitutions can be made and tested usingknown methods of mutagenesis, recombination, and/or shuffling, followedby a relevant screening procedure, such as those disclosed byReidhaar-Olson and Sauer, 1988, Science 241: 53-57; Bowie and Sauer,1989, Proc. Natl. Aced. Sci. USA 86: 2152-2156; WO 95/17413; or WO95/22625. Other methods that can be used include error-prone PCR, phagedisplay (e.g., Lowman et al., 1991, Biochem. 30:10832-10837; U.S. Pat.No. 5,223,409; WO 92/06204), and region-directed mutagenesis (Derbyshireet al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

An N-terminal extension of the polypeptides of the invention maysuitably consist of from 1 to 50 amino acids, preferably 2-20 aminoacids, especially 3-15 amino acids. In one embodiment N-terminal peptideextension does not contain an Arg (R). In another embodiment theN-terminal extension comprises a kex2 or kex2-like cleavage site as willbe defined further below. In a preferred embodiment the N-terminalextension is a peptide, comprising at least two Glu (E) and/or Asp (D)amino acid residues, such as an N-terminal extension comprising one ofthe following sequences: EAE, EE, DE and DD.

Methods and Uses

Human beta defensin 2 was found to significantly reduce the severity ofdisease parameters in a 10-Day Dextran Sodium Sulphate (DSS)-inducedcolitis model in the mouse; thus showing potent activity as a medicamentfor treatment of inflammatory bowel diseases, such as ulcerative colitisand Chrohn's disease.

The present invention therefore provides methods of treatinginflammatory bowel diseases, which treatment comprises administeringparenterally to a subject in need of such treatment an effective amountof a mammal beta defensin, such as human beta defensin 2, e.g., in theform of a pharmaceutical composition. Also provided are mammal betadefensins, such as human beta defensin 2, for the manufacture of amedicament for parenteral administration, and the use of mammal betadefensins, such as human beta defensin 2, for the manufacture of amedicament for parenteral administration, e.g., a pharmaceuticalcomposition, for the treatment of inflammatory bowel disease. Treatmentincludes treatment of an existing disease or disorder, as well asprophylaxis (prevention) of a disease or disorder.

In an embodiment, the treatment results in reduced TNF-alpha activity intreated tissues, preferably reduced TNF-alpha activity and increasedIL-10 activity.

Mammal beta defensins can be employed therapeutically in compositionsformulated for administration by any conventional route, includingenterally (e.g., buccal, oral, nasal, rectal), parenterally (e.g.,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular), or topically (e.g., epicutaneous, intranasal, orintratracheal). Within other embodiments, the compositions describedherein may be administered as part of a sustained release implant.

Within yet other embodiments, compositions, of preferred embodiments maybe formulized as a lyophilizate, utilizing appropriate excipients thatprovide stability as a lyophilizate, and subsequent to rehydration.

Pharmaceutical compositions containing a mammal beta defensin, such as ahuman beta defensin, can be manufactured according to conventionalmethods, e.g., by mixing, granulating, coating, dissolving orlyophilizing processes.

Pharmaceutical compositions of preferred embodiments comprise a mammal adefensin, such as a human beta defensin, and a pharmaceuticallyacceptable carrier and/or diluent.

A mammal beta defensin, such as a human beta defensin, is preferablyemployed in pharmaceutical compositions in an amount which is effectiveto treat an inflammatory bowel disease, preferably with acceptabletoxicity to the patient. For such treatment, the appropriate dosagewill, of course, vary depending upon, for example, the chemical natureand the pharmacokinetic data of a compound of the present inventionused, the individual host, the mode of administration and the nature andseverity of the conditions being treated. However, in general, forsatisfactory results in larger mammals, for example humans, an indicateddaily dosage is preferably from about 0.001 g to about 1.5 g, morepreferably from about 0.01 g to 1.0 g; or from about 0.001 mg/kg bodyweight to about 20 mg/kg body weight, preferably from about 0.01 mg/kgbody weight to about 20 mg/kg body weight, more preferably from about0.1 mg/kg body weight to about 10 mg/kg body weight, for example,administered in divided doses up to one, two, three, or four times aday. The compounds of preferred embodiments can be administered tolarger mammals, for example humans, by similar modes of administrationat similar dosages than conventionally used.

In certain embodiments, the pharmaceutical compositions of preferredembodiments can include a mammal beta defensin, such as a human betadefensin, in an amount of about 0.5 mg or less to about 1500 mg or moreper unit dosage form depending upon the route of administration,preferably from about 0.5, 0.6, 0.7, 0.8, or 0.9 mg to about 150, 200,250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 mg, and morepreferably from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 mg toabout 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mg.In certain embodiments, however, lower or higher dosages than thosementioned above may be preferred. Appropriate concentrations and dosagescan be readily determined by one skilled in the art.

Pharmaceutically acceptable carriers and/or diluents are familiar tothose skilled in the art. For compositions formulated as liquidsolutions, acceptable carriers and/or diluents include saline andsterile water, and may optionally include antioxidants, buffers,bacteriostats, and other common additives. The compositions can also beformulated as pills, capsules, granules, tablets (coated or uncoated),(injectable) solutions, solid solutions, suspensions, dispersions, soliddispersions (e.g., in the form of ampoules, vials, creams, gels, pastes,inhaler powder, foams, tinctures, lipsticks, drops, sprays, orsuppositories). The formulation can contain (in addition to a mammalbeta defensin, and other optional active ingredients) carriers, fillers,disintegrators, flow conditioners, sugars and sweeteners, fragrances,preservatives, stabilizers, wetting agents, emulsifiers, solubilizers,salts for regulating osmotic pressure, buffers, diluents, dispersing andsurface-active agents, binders, lubricants, and/or other pharmaceuticalexcipients as are known in the art. One skilled in this art may furtherformulate mammal beta defensins in an appropriate manner, and inaccordance with accepted practices, such as those described inRemington's Pharmaceutical Sciences, Gennaro, Ed., Mack Publishing Co.,Easton, Pa. 1990.

A mammal beta defensin, such as a human beta defensin, can be usedalone, or in combination therapies with one, two, or more otherpharmaceutical compounds or drug substances, and/or with one or morepharmaceutically acceptable excipient(s).

In Vitro Synthesis

Mammal beta defensins may be prepared by in vitro synthesis, usingconventional methods as known in the art. Various commercial syntheticapparatuses are available, for example automated synthesizers by AppliedBiosystems Inc., Beckman, etc. By using synthesizers, naturallyoccurring amino acids may be substituted with unnatural amino acids,particularly D-isomers (or D-forms) e.g. D-alanine and D-isoleucine,diastereoisomers, side chains having different lengths orfunctionalities, and the like. The particular sequence and the manner ofpreparation will be determined by convenience, economics, purityrequired, and the like.

Chemical linking may be provided to various peptides or proteinscomprising convenient functionalities for bonding, such as amino groupsfor amide or substituted amine formation, e.g. reductive amination,thiol groups for thioether or disulfide formation, carboxyl groups foramide formation, and the like.

If desired, various groups may be introduced into the peptide duringsynthesis or during expression, which allow for linking to othermolecules or to a surface. Thus cysteines can be used to makethioethers, histidines for linking to a metal ion complex, carboxylgroups for forming amides or esters, amino groups for forming amides,and the like.

Mammal beta defensins, or functional equivalents thereof, may also beisolated and purified in accordance with conventional methods ofrecombinant synthesis. A lysate may be prepared of the expression hostand the lysate purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, or other purificationtechnique.

The present invention is further described by the following examplesthat should not be construed as limiting the scope of the invention.

EXAMPLES

During testing of hBD2 for immunomodulatory effects it was unexpectedlyobserved that hBD2 had vast anti-inflammatory potential.

Here, we have shown that hBD2 has significant effect in treatinginflammatory bowel disease (colitis) induced by oral dextran sodiumsulphate (DSS) administration in the mouse. We have also shown that hBD2has downregulating potential on TNF-alpha.

Example 1

Production of Human Beta Defensin 2 (hBD2)

hBD2 was produced recombinantly. A synthetic DNA fragment (DNA 2.0)encoding hBD2 was cloned into the pET-32(+) expression vector (Novagen).The resulting plasmid encoded a translational fusion peptide containingan N-terminal thioredoxin part followed by a his-tag, an enterokinasecleavage site and finally the hBD2 peptide. The expression plasmid wastransformed into E. coli strain BL21.

An overnight culture of this strain was diluted 100 fold in TB-glycerolcontaining 100 μg/ml of ampicillin and grown to an OD600 ofapproximately 8 at 37° C. and induced with 0.5 mM of IPTG for 3 hoursafter which the cells were harvested by centrifugation. The his-taggedtrx-hBD2 fusion peptide was purified on Ni-NTA beads (QIAGEN) usingstandard protocols. The his-tag purified fusion peptide was subsequentlydialysed over-night into enterokinase buffer (50 mM tris-HCl pH 7.5, 1mM CaCl₂) and cleaved with enterokinase to release mature hBD2. The hBD2peptide was further purified by cation-exchange chromatography usingSource 15 S matrix (Amersham Biosciences). The correct molecular weightof hBD2 was verified using MALDI-TOF mass spectrometry.

Production of mBD3 (see Example 7) was carried out using an identicalprotocol.

The proper folding and disulphide-bridge topology of the hBD2 moleculewas subsequently verified using tryptic digestion coupled with LC-MS andNMR spectroscopy.

Endotoxin was removed by preparative RP-HPLC at low pH, and the contentof endotoxin was determined by a LAL assay (Endosafe KTA2) and the levelwas found to be below the detection limit of the assay (0.05 EU/mg). Toascertain that levels below the detection limit of the endotoxin assaywere not able to stimulate PBMC, titration curves of stimulation with avery potent lipopolysaccharide (E. coli, O111:B4, Sigma L4391) wereperformed. Very low levels of this LPS (0.06 ng/ml) were able tostimulate PBMC to a detectable cytokine production.

Example 2 10-Day Dextran Sodium Sulphate (DSS)-Induced Colitis Model inthe Mouse

The aim of the following study was to determine the anti-inflammatoryactivity of human beta defensin 2 in an acute (10-days) model ofinflammatory bowel disease (colitis) induced by oral dextran sodiumsulphate (DSS) administration in the mouse.

The DSS colitis mouse model is a well recognized model for studyinginflammatory bowel disease, as described in Kawada et al., “Insightsfrom advances in research of chemically induced experimental models ofhuman inflammatory bowel disease”, World J. Gastroenterol. 13(42):5581-5593 (2007); and Wirtz and Neurath, “Mouse models of inflammatorybowel disease”, Advanced Drug Delivery Reviews 59(11): 1073-1083 (2007).

Materials Test Items

Human beta defensin 2 (hBD2); see Example 1 aboveMethylprednisolone 21-hemisuccinate (“prednisolone”)PBS buffer (GIBCO)

Experimental Animals

Male C57BL/6 mice (Harlan Interfauna Ibérica, Barcelona, Spain) wereused in the study, as this is a species and sex that has beendemonstrated to develop significant inflammation of the colon whenadministered a 2% solution of DSS in the drinking water over a period of10 days.

Identification

Animals were identified by number and letter codes on theft tails.Additionally, each cage was identified by a color-coded card indicatingthe number and sex of the animals, the test item code or name, doselevel, administration route, treatment period, group number, study codeand study director's name.

Weight

The average body weight of the animals on the day of start of the studywas 22.4±0.16 g.

Acclimatization (Quarantine)

Minimum of 7 days prior to the start of the study, under the sameconditions as those of the main study.

Housing

On arrival, the animals were separated and housed at random inpolycarbonate cages (E-Type, Charles River, 255×405×197 mm) withstainless steel lids.

Animals were housed in groups of five animals per cage according totheir sex, in animal rooms with controlled temperature (22±2° C.),lighting (12/12 hours light/darkness), air pressure, number of airrenovations and relative humidity (30-70%).

The cages all had sawdust (Lignocel 3-4; Harlan Interfauna Ibérica,Spain) on the floor as litter.

Food and Water

All mice had free access to a dry, pelleted standard rodent diet (TekladGlobal 2014; Harlan Interfauna Ibérica, Spain).

Water was provided ad libitum in bottles. Tap water supply to the animalrooms is periodically analyzed to check its composition and to detectpossible contaminants (chemical and microbiological).

Equipment and Materials Equipment:

Animal balance Sartorius Mod. BP 2100

Surgical dissection equipment

Eppendorf 54150 centrifuge

Nikon Eclipse E600FN microscope

Hook & Tucker instruments rotamixer

IKA UltraTurrax Homogeniser

Sartorius Mod. BP 221S analytical balance

ELISA microplate reader Labsystems Multiskan EX

Materials and Reagents:

Sterile disposable syringes (1 ml)

Sterile Butterfly 25 G infusion set

Anaesthetic (Ketamine/Xylazine)

Topical Anaesthetic cream (EMLA, Astra Zeneca)

Dextran Sodium Sulphate 30.000-50.000 Da (MP Biomedicals)

Phosphate Buffered Saline (PBS; Sigma)

Neutral Buffered Formalin (VWR)

Bovine Serum Albumin (Sigma)

Protease Inhibitor Cocktail (Sigma)

Mouse TNF-α ELISA kit (GE Healthcare)

Experimental Protocol Study Design

Animals were divided into 5 experimental groups. Each group consisted of10 males:

Group A: Treated with Control vehicle (PBS) i.v.Group B: Treated with hBD2 (0.1 mg/kg i.v.)Group C: Treated with hBD2 (1 mg/kg i.v.)Group D: Treated with hBD2 (10 mg/kg i.v.)Group E: Treated with methylprednisolone (1 mg/kg p.o.)

Animal allocation to all experimental groups was done in a randomizedmanner. A maximum of 5 mice were housed in each cage (as per Directive86/609/EEC). All animals were weighed on their arrival at the laboratoryand prior to the administration of the test items.

Administration of the Test Substance

The control vehicle and hBD2 were administered intravenously via thetail vein with the use of a sterile needle (25 G) in a dosing volume of5 ml/kg body weight as a slow bolus. The animals received one dose daily(every 24 hours) of the corresponding test item (hBD2, prednisolone orcontrol vehicle) for 10 consecutive days.

Prednisolone was given orally at a dose of 1 mg/kg in a dosing volume of5 ml/kg body weight, in the same dosing regime as hBD2.

Experimental Procedure Induction of Colitis

Colitis was induced in mice by supplementing their drinking water withDSS 2% for 7 days.

On Day 1 all mice were weighed and marked according to theirexperimental groups. The drinking bottle of each cage was filled withthe DSS solution, making sure all bottle lids were mounted properly andthat none were congested.

On Day 3 any remaining solution in the bottles was emptied and refilledwith fresh DSS solution. This procedure was repeated again on Day 5.

On Day 8 any remaining solution was discarded and replaced withautoclaved water.

Animals were sacrificed 2 days later on Day 10.

Clinical Assessment (Disease Activity Index)

Daily clinical assessment of DSS-treated animals was carried out, withthe calculation of a validated clinical Disease Activity Index (DAI)ranging from 0 to 4 according to the following parameters: stoolconsistency, presence or absence of rectal bleeding and weight loss:

Parameter DAI score Change in Body Weight:   <1% 0  1-5% 1  5-10% 210-15% 3   >15% 4 Rectal Bleeding: Negative 0 Positive 4 StoolConsistency: Normal 0 Loose Stools 2 Diarrhoea 4

Bodyweight loss was calculated as the percent difference between theoriginal bodyweight (Day 1) and the actual bodyweight on eachexperimental day (2-10).

The appearance of diarrhoea is defined as mucus/faecal material adherentto anal fur. Rectal bleeding is defined as diarrhoea containing visibleblood/mucus or gross rectal bleeding. The maximum score of the DAI eachday is 12.

Blood Sampling

Two blood samples were obtained from each animal on two separateoccasions during the course of the study: on Day 1 and on Day 5. Bloodsamples were obtained on each occasion into Microvette CB-300 microtubesby puncture of the saphenous vein 2 hours after administration of thetest item. This blood extraction method does not require anaesthetic oranalgesics and produces a minimum stress in the animals (Hem et al.,1998). Additionally a terminal blood sample was obtained from allanimals on the last day of the study from the abdominal vena cave alsotwo hours after test item administration.

Blood samples were allowed to clot and then centrifuged at 3000 rpm for10 min and the resulting serum frozen at −80° C. for storage.

Euthanasia and Collection of Colon Samples

On day 10, two hours after the last administration of control vehicle,hBD2 or prednisolone, the animals were killed by an overdose ofanaesthetic. Their colons were removed and their length and weightmeasured after exclusion of the caecum.

Two sections (proximal and distal) of colon were taken from each animaland preserved in neutral buffered formalin for subsequent histologicalanalysis (haematoxylin and eosin staining) according to the followingscoring system:

Description Score No changes observed 0 Minimal scattered mucosalinflammatory cell infiltrates, 1 with or without minimal epithelialhyperplasia. Mild scattered to diffuse inflammatory cell infiltrates, 2sometimes extending into the submucosa and associated with erosions,with minimal to mild epithelial hyperplasia and minimal to mild mucindepletion from goblet cells. Mild to moderate inflammatory cellinfiltrates that were 3 sometimes transmural, often associated withulceration, with moderate epithelial hyperplasia and mucin depletion.Marked inflammatory cell infiltrates that were often 4 transmural andassociated with ulceration, with marked epithelial hyperplasia and mucindepletion. Marked transmural inflammation with severe ulceration and 5loss of intestinal glands.

Determination of TNF-Alpha Concentration in Colonic Tissue Samples

An additional sample of colon was obtained from each animal andhomogenised in PBS (100 mg tissue/ml PBS) containing 1% bovine serumalbumin (BSA) and a protease inhibitor cocktail (1 ml/20 g tissue). Thehomogenate was then be centrifuged at 1400 rpm for 10 min and thesupernatant stored at −20° C. for subsequent determination of TNF-αconcentration by specific enzyme immunoassay (ELISA).

Results Disease Activity Index Score

TABLE 1 Disease Activity Index (DAI) score progression during Day 1 toDay 10. DAI score Test item Data Day 1 Day 2 Day 3 Day 4 Day 5 Group AMean 0.00 1.10 1.30 3.20 2.90 Control S.E.M. 0.00 0.31 0.37 0.36 0.31vehicle i.v. Group B Mean 0.00 0.20 0.80 2.90 2.80 hBD2 S.E.M. 0.00 0.130.20 0.10 0.13 0.1 mg/kg i.v. Group C Mean 0.00 0.00 0.22 2.22 2.44 hBD2S.E.M. 0.00 0.00 0.22 0.15 0.18 1 mg/kg i.v. Group D Mean 0.00 0.60 1.003.67 3.11 hBD2 S.E.M. 0.00 0.22 0.44 0.24 0.26 10 mg/kg i.v. Group EMean 0.00 0.10 0.00 2.60 2.50 Prednisolone S.E.M. 0.00 0.10 0.00 0.220.22 1 mg/kg p.o. DAI score Test item Data Day 6 Day 7 Day 8 Day 9 Day10 Group A Mean 3.10 4.10 5.90 8.90 10.90 Control S.E.M. 0.31 0.69 1.261.02 0.62 vehicle i.v. Group B Mean 3.20 1.44** 2.11* 3.89** 6.44* hBD2S.E.M. 0.20 0.38 0.20 0.35 0.85 0.1 mg/kg i.v. Group C Mean 2.89 2.223.67 5.22 6.44* hBD2 S.E.M. 0.20 0.43 0.80 0.83 1.08 1 mg/kg i.v. GroupD Mean 3.22 2.11 4.11 6.78 7.33 hBD2 S.E.M. 0.28 0.31 0.93 1.20 1.33 10mg/kg i.v. Group E Mean 2.60 3.10 2.50* 3.80* 4.90** Prednisolone S.E.M.0.27 0.96 0.43 0.98 0.91 1 mg/kg p.o. Significant differences fromcontrol (vehicle) group values at a given date are shown as *p < 0.05;**p < 0.01 (Kruskal-Wallis Test for non-parametric data).

Histological Evaluation

Two sections (proximal and distal) of colon were taken from each animal,processed for histological analysis (haematoxylin and eosin staining)and scored by a blind observer according to the histological scoringsystem described above.

Determination of TNF-α Concentration in Colonic Tissue

An additional sample of colon was obtained from each animal andhomogenised in PBS (100 mg tissue/ml PBS) containing 1% bovine serumalbumin (BSA) and a protease inhibitor cocktail (1 ml/20 g tissue). Thehomogenate was then be centrifuged at 14000 rpm for 10 min and thesupernatant stored at −20° C. for subsequent determination of TNF-αconcentration by specific enzyme immunoassay (ELISA).

TABLE 3 Histological scores, colon weight and length, and colon TNF-αconcentration. Histology Histology Colon TNF-α Score Score concentrationTest item Data Proximal Colon Distal Colon (pg/g tissue) Group A Mean4.20 4.50 1864 Control S.E.M. 0.25 0.22 227 vehicle i.v. Group B Mean2.22** 3.87 1185 hBD2 S.E.M. 0.43 0.47 205 0.1 mg/kg i.v. Group C Mean2.89* 4.13 1457 hBD2 S.E.M. 0.35 0.35 211 1 mg/kg i.v. Group D Mean2.89* 4.78 1212 hBD2 S.E.M. 0.39 0.15 211 10 mg/kg i.v. Group E Mean2.80* 3.70 1833 Prednisolone S.E.M. 0.51 0.42 414 1 mg/kg p.o.Differences in histological scores from control (vehicle) group valuesare shown as *p < 0.05; **p < 0.01 (Kruskal-Wallis Test fornon-parametric data).

Statistical Analysis

The statistical significance of the results was evaluated using thestatistics program Graphpad Instat 3. The difference between groups fordisease activity index and histological score was evaluated byKruskal-Wallis test for unpaired data plus post-test Dunn to allow formultiple comparisons. A value of p<0.05 was taken as significant.

Conclusions

The results demonstrate that hBD2 at the lowest dose tested (0.1 mg/kgi.v.) significantly reduces the increase in Disease Activity Indexinduced by DSS administration at day 7 (1.44±0.38 test item vs. 4.1±0.69vehicle; p<0.01), day 8 (2.11±0.2 test item vs. 5.9±1.26 vehicle;p<0.05), day 9 (3.89±0.35 test item vs. 8.9±1.02 vehicle; p<0.01) andday 10 (6.44±0.85 test item vs. 10.9±0.62 vehicle; p<0.05).

Treatment with the intermediate dose of hBD2 (1 mg/kg i.v.) for 10consecutive days resulted in an apparent reduction of the diseaseactivity index score but this was only significant on day 10 (6.44±1.08test item vs. 10.9±0.62 vehicle; p<0.05).

Similarly to the results obtained with the Disease Activity Index on day10, histological analysis of the proximal colons of each animal revealeda very significant reduction of histological damage score by treatmentwith the low dose of hBD2 (2.22±0.43 test item vs. 4.2±0.25 vehicle;p<0.01). Moreover, a significant reduction of histological injury wasalso observed with the intermediate and high doses of hBD2, as well aswith prednisolone (2.89%0.35; 2.89±0.39 and 2.8±0.5 respectively;p<0.05). In contrast, in the distal colon—although an apparent reductionin histological injury could be observed in the animals treated with thelow and intermediate dose of hBD2, as well as with prednisolone—this wasnot statistically significant. No reduction could be observed in theanimals that were treated with the high dose of hBD2.

Similarly, treatment with the low and intermediate doses of hBD2resulted in an apparent reduction in colonic TNF-alpha levels, but thisapparent reduction was not statistically significant.

The results obtained in the present study demonstrate ananti-inflammatory activity of hBD2 in the model of DSS colitis inducedin the mouse after a 10-day treatment period. However, thisanti-inflammatory activity appears to be more pronounced at the lowerdose of hBD2 used (0.1 mg/kg/day i.v.) and is gradually lost withincreasing doses up to the highest dose used in the study (10 mg/kg/dayi.v.). Moreover, the anti-inflammatory effect of the lowest dose of hBD2is comparable or even greater (e.g. histological score) than that ofprednisolone at a dose of 1 mg/kg/day p.o.

Example 3 10-Day Dextran Sodium Sulphate (DSS)-Induced Colitis Model inthe Mouse

Example 3 was carried out essentially as described in Example 2. Thedifferences are indicated below.

Weight

The average body weight of the animals on the day of start of the studywas 19.74±0.09 g (mean±SEM).

Study Design

Animals were divided into 9 experimental groups. Each group consisted of10 males:

Group A: Treated with control vehicle (PBS) i.v.Group B: Treated with hBD2 (1 mg/kg i.v.)—once dailyGroup C: Treated with hBD2 (0.1 mg/kg i.v.)—once dailyGroup D: Treated with hBD2 (0.01 mg/kg i.v.)—once dailyGroup E: Treated with hBD2 (0,001 mg/kg i.v.)—once dailyGroup F: Treated with hBD2 (0.1 mg/kg i.v. s.c.)—twice dailyGroup G: Treated with hBD2 (0.1 mg/kg i.v.)—every second dayGroup H: Treated with methylprednisolone (1 mg/kg p.o.)Group J: Treated with methylprednisolone (10 mg/kg p.o.)

Animal allocation to all experimental groups was done in a randomizedmanner. A maximum of 5 mice were housed in each cage (as per Directive86/609/EEC). All animals were weighed on their arrival at the laboratoryand prior to the administration of the test and reference compounds.

Administration of the Test Items

The control vehicle and hBD2 were administered intravenously via thetail vein with the use of a sterile needle (25 G) in a dosing volume of5 ml/kg body weight as a slow bolus (over a period of 15 seconds).

The animals in groups A to E received one dose daily (every 24 hours) ofthe corresponding test item (hBD2, prednisolone or control vehicle) for10 consecutive days.

The animals in group F received one dose i.v. and another dose s.c. (12hours after the i.v. dose) of the corresponding test item for 10consecutive days.

The animals in group G received one dose every two days of thecorresponding test item for 10 consecutive days.

Methylprednisolone was given orally at a dose of 1 mg/kg (group H) and10 mg/kg (group J) in a dosing volume of 5 ml/kg body weight, once dailyfor 10 consecutive days.

Blood Sampling

A terminal blood sample was obtained from all animals on the last day ofthe study from the abdominal vena cava 2 hours after test itemadministration.

Blood samples were allowed to clot and then centrifuged at 3000 rpm for10 min, and the resulting serum was frozen at −80° C. for subsequentanalysis.

Results Disease Activity Index Score

TABLE 4 Disease Activity Index (DAI) score progression during Day 1 toDay 10. DAI score Test item Data Day 1 Day 2 Day 3 Day 4 Day 5 Group AMean 0.00 0.00 0.10 0.10 0.20 Control S.E.M. 0.00 0.00 0.10 0.10 0.13vehicle i.v. Group B Mean 0.00 0.10 0.20 0.40 0.30 hBD2 S.E.M. 0.00 0.100.13 0.16 0.21 1 mg/kg i.v. Group C Mean 0.00 0.44 0.89 0.56 0.78 hBD2S.E.M. 0.00 0.18 0.42 0.29 0.28 0.1 mg/kg i.v. Group D Mean 0.00 0.000.30 0.40 1.60 hBD2 S.E.M. 0.00 0.00 0.15 0.16 0.43 0.01 mg/kg i.v.Group E Mean 0.00 0.00 0.10 0.20 0.40 hBD2 S.E.M. 0.00 0.00 0.10 0.130.16 0.001 mg/kg i.v. Group F Mean 0.00 0.30 0.70 0.70 0.60 hBD2 S.E.M.0.00 0.21 0.30 0.34 0.16 0.1 mg/kg twice daily i.v. + s.c. Group G Mean0.00 0.20 0.40 0.50 0.50 hBD2 S.E.M. 0.00 0.13 0.22 0.17 0.17 0.1 mg/kgi.v. every 2. day Group H Mean 0.00 0.50 0.50 0.40 1.10 PrednisoloneS.E.M. 0.00 0.17 0.17 0.16 0.18 1 mg/kg p.o. Group J Mean 0.00 0.30 0.700.80 1.30 Prednisolone S.E.M. 0.00 0.15 0.21 0.20 0.21 10 mg/kg p.o. DAIscore Test item Data Day 6 Day 7 Day 8 Day 9 Day 10 Group A Mean 6.909.67 11.11 11.67 11.00 Control S.E.M. 1.02 0.33 0.31 0.17 0.65 vehiclei.v. Group B Mean 2.30* 4.40* 6.89 5.00* 5.78* hBD2 S.E.M. 1.00 1.031.41 0.60 0.70 1 mg/kg i.v. Group C Mean 1.56** 4.13* 5.43* 6.29* 6.86hBD2 S.E.M. 0.73 0.83 1.13 1.64 1.14 0.1 mg/kg i.v. Group D Mean 2.706.50 6.20* 4.60*** 5.20** hBD2 S.E.M. 1.08 1.28 1.06 0.98 0.87 0.01mg/kg i.v. Group E Mean 3.40 7.11 8.56 5.89** 6.67 hBD2 S.E.M. 1.32 1.381.06 1.63 1.30 0.001 mg/kg i.v. Group F Mean 0.70*** 3.50** 4.00***2.90*** 4.50*** hBD2 S.E.M. 0.30 0.89 1.17 0.55 0.62 0.1 mg/kg twicedaily i.v. + s.c. Group G Mean 2.90 6.50 8.70 7.50 6.56 hBD2 S.E.M. 1.121.11 1.25 0.93 0.99 0.1 mg/kg i.v. every 2. day Group H Mean 3.80 5.906.40 5.60* 5.60* Prednisolone S.E.M. 0.98 1.16 0.88 0.88 0.65 1 mg/kgp.o. Group J Mean 2.00 3.20** 4.80** 5.20* 4.00*** Prednisolone S.E.M.0.30 0.73 0.53 0.61 0.00 10 mg/kg p.o. Significant differences fromcontrol (vehicle) group values at a given date are shown as *p < 0.05;**p < 0.01 (Kruskal-Wallis Test for non-parametric data). Day 6 to Day10 is shown on the next page.

Histological Evaluation

Two sections (proximal and distal) of colon were taken from each animal,processed for histological analysis (haematoxylin and eosin staining),and scored by a blind observer according to the scoring system describedabove.

TABLE 5 Histological scores, colon weight and length, and colon TNF-αconcentration. Histology Histology Score Score Test item Data ProximalColon Distal Colon Group A Mean 2.44 4.87 Control vehicle i.v. S.E.M.0.34 0.17 Group B Mean 1.78 3.56 hBD2 S.E.M. 0.36 0.38 1 mg/kg i.v.Group C Mean 1.71 3.14* hBD2 S.E.M. 0.18 0.40 0.1 mg/kg i.v. Group DMean 1.70 3.10** hBD2 S.E.M. 0.26 0.23 0.01 mg/kg i.v. Group E Mean 1.443.58 hBD2 S.E.M. 0.24 0.18 0.001 mg/kg i.v. Group F Mean 1.30* 2.90***hBD2 S.E.M. 0.21 0.23 0.1 mg/kg twice daily i.v. + s.c. Group G Mean1.56 3.58 hBD2 S.E.M. 0.24 0.29 0.1 mg/kg i.v. every 2. day Group H Mean1.40 3.00*** Prednisolone S.E.M. 0.22 0.00 1 mg/kg p.o. Group J Mean1.40 2.70*** Prednisolone S.E.M. 0.16 0.21 10 mg/kg p.o. Differences inhistological scores from control (vehicle) group values are shown as *p< 0.05; **p < 0.01 (Kruskal-Wallis Test for non-parametric data).

Statistical Analysis

The statistical significance of the results was evaluated using thestatistics program Graphpad Instat 3. The difference between groups fordisease activity index and histological score was evaluated byKruskal-Wallis test for unpaired data+post-test of Dunn for multiplecomparisons. A value of p<0.05 was taken as significant. In the tablesabove, significant differences versus the corresponding control(vehicle) group are denoted as: *p<0.05, **p<0.01, ***p<0.001.

Conclusions

The aim of the present study was to determine the anti-inflammatoryactivity of hBD2 in an acute (10-days) model of inflammatory boweldisease (colitis) induced by oral dextran sodium sulphate (DSS, 2%)administration in the mouse.

The results obtained in the present study further demonstrate ananti-inflammatory activity of hBD2 in the model of DSS colitis inducedin the mouse after a 10-day treatment period.

This anti-inflammatory activity appears to be more pronounced afteradministration of hBD2 twice per day (every 12 hours), bothintravenously and subcutaneously, at a dose of 0.1 mg/kg.

Moreover, the anti-inflammatory effect observed with this dose of hBD2is comparable, or even greater (both on Disease Activity Index andhistological score), than that of prednisolone at a dose of 1 mg/kg or10 mg/kg given orally.

Example 4

Anti-Inflammatory Activity of Human Beta Defensin 2 (hBD2)

In human PBMC cultures it was observed that treatment with hBD2 hadgreat influence on the cytokine profile of LPS, LTA or peptidoglycanstimulated cultures. It has previously been observed that hBD2 is ableto induce the proinflammatory cytokines and chemokines IL-6, IL-18,RANTES, IP-10 and IL-8 (Niyonsaba et al. 2007, Boniotto M. et al. 2006).

Here we show that hBD2 has downregulating potential on TNF and IL-1β,two proinflammatory cytokines; and hBD2 also induces IL-10 uponinduction of an inflammatory stimulus with lipopolysaccharide (LPS),lipoteichoic acid (LTA) or peptidoglycan (PGN). IL-10 is a potentialanti-inflammatory cytokine and hence the resulting effect of hBD2 isanti-inflammatory. This has been observed for human PBMC, a monocyticcell line and a dendritoid cell line.

hBD2 was prepared as described in Example 1,

Isolation and Stimulation of PBMC

Peripheral blood was drawn from healthy volunteers (with approval fromthe relevant ethical committee in Denmark). Heparinized blood wasdiluted 1/1 v/v with RPMI and were subjected to Ficoll densitycentrifugation within 2 h of drawing. Plasma was collected from the topfrom individual donors and was kept on ice until it was used at 2% inthe culture medium (autologous culture medium). Isolated PBMC wereresuspended in autologous culture medium and seeded in 96-well cultureplates with 255.000 cells per well in a total of 200 μl. PBMC from thesame donor were stimulated with 100, 10 or 1 μg/ml of hBD2 either aloneor together with LPS at 0.6 ng/ml or 20 ng/ml (E. coli, O111:B4, SigmaL4391), Lipoteichoic acid (LTA) at 1.25 μg/ml (from B. subtilis, SigmaL3265) or peptidoglycan (PGN) at 40 μg/ml (from S. aureus, Sigma 77140).The concentrations used for stimulation were optimized on 3 differentdonors in initial experiments, for LPS two different concentrations wereused to be sure to be on a cytokine level that is possible to modulate.In some experiments PBMC were treated with Dexamethason and Indomethacinalone and together with LPS or LTA as a control on downregulation ofinflammatory cytokines. The supernatants were collected after incubationat 37° C. for 24 hours, and stored at −80° C. until cytokinemeasurement. Viability was measured by Alamar Blue (Biosource, DALL1100) in all experiments and in some cases also by MTS (Promega)according to manufacturer's instruction and was in some experiments alsojudged by counting of the cells by a Nucleocounter.

Culture and Stimulation of MUTZ-3

The human myeloid leukaemia-derived cell line MUTZ-3 (DSMZ,Braunschweig, Germany) was maintained in a-MEM (Sigma M4526),supplemented with 20% [volume/volume (v/v)] fetal bovine serum (SigmaF6178) and 40 ng/ml rhGM-CSF (R&D Systems 215-GM-050). These progenitorcells is in the following denoted monocyte cell line and these monocyteswere stimulated with 100, 10 or 1 μg/ml of hBD2 either alone or togetherwith LPS or LTA.

Dendritic Cell Differentiation

To generate a dendritoid cell line, the human myeloid leukaemia celllines MUTZ-3 (1×10⁵ cells/ml) was differentiated for 7 days in thepresence of rhGM-CSF (150 ng/ml) and rhIL-4 (50 ng/ml) into immatureDCs. Medium was exchanged every 2-3 days. The differentiated cell linewas further stimulated with either LPS or LTA with and without hBD2 toexplore the effect of hBD2 on dendritic cells.

Cytokine Measurements

Cytokine production in supernatants was measured by flow cytometry witha human inflammation cytometric bead array (CBA) according tomanufacturer's instructions (BD) on a FACSarray flow cytometer. Thefollowing cytokines were measured: IL-8, IL-β, IL-10, TNF, 12 p70, IL-6.In some experiments, cytokines were measured by ELISA kits from R&Dsystems (IL-10, TNF-α, IL-1β) according to the manufacturer'instruction.

Data Analysis

All experiments were performed at least twice, with representativeresults shown. The data presented are expressed as mean plus/minusstandard deviation (SD). Statistical significance was determined by2-way ANOVA with the variables being treatment (hBD2, dexamethazone,etc.) and stimulation (LPS, LTA, peptidoglycan, etc.) followed byBonferroni post-test as reported in the table legends. Differences wereconsidered significant for p<0.05.

Results

The effect of hBD2 was tested on human PBMC treated with and without LPSand LTA (Tables 6, 7 and 8). Treatment with hBD2 gave a significantdownregulation of TNF in stimulated cultures for all three testedconcentrations (Table 6), the downregulation is dose-dependent for LPSat 0.6 ng/ml and for LTA. For IL-1β the downregulation was observedmostly at the highest doses (Table 7). Interestingly, IL-10 wassignificantly and dose-dependently upregulated (Table 8). Downregulationof proinflammatory cytokines and induction of anti-inflammatorycytokines shows a very strong anti-inflammatory potential of hBD2.Viability was measured by two different assays, in order to exclude thatthe anti-inflammatory effects of hBD2 is due to cytotoxic effects. InTables 9 and 10 it can be seen that hBD2 have no cytotoxic effect on thecells, the observed effects are stimulatory effects due to stimulationwith LPS or LTA that leads to proliferation of the cells. Therefore hBD2has no cytotoxic effect on these cells.

In Tables 11, 12 and 13, supernatants from another donor were analyzedfor cytokines by ELISA instead of by a cytometric bead array byflowcytometry and here the same were observed, although the sensitivityof the assay is lower and the detection limit much higher and thereforethe effects were not as significant.

In order to test yet another Toll-like receptor ligand, the effect ofhBD2 on peptidoglycan stimulated PBMC was investigated (Tables 14 and15). The same was observed: TNF is dose-dependently downregulated andIL-10 is dose-dependently induced.

As a positive control on downregulation of TNF, two anti-inflammatorycompounds, dexamethasone and Indomethacin, were tested in the assay. Theconcentrations are selected so the compounds are not toxic andachievable concentration due to solubility in medium. Indomethacin onlyinhibited TNF (Table 16) after stimulation with LTA, whereasdexamethasone effectively downregulated TNF production, the same wasobserved for IL-1β (Table 18). Indomethacin is a COX-1 and COX-2inhibitor and is a nonsteroidal anti-inflammatory drug (NSAID) used totreat mild to moderate pain and help relieve symptoms of arthritis anddexamethasone is a synthetic glucocorticoid used primarily in thetreatment of inflammatory disorders and it has very potentdownregulating effect on proinflammatory cytokines (Rowland et al. 1998)at very low doses, which we also observe for TNF-α and IL-1β. hBD2 is aseffective as or better than these two anti-inflammatory compounds.

In Tables 19 and 20, the effect of hBD2 on downregulating TNF in amonocyte cell line and on dendritic cells are shown, the same isobserved as was for PBMC. IL-10 was also induced for dendritic cellsstimulated with hBD2 and LPG or hBD2 and LTA (results not shown).

In order to exclude that binding of hBD2 to LPS or LTA causes thedownregulation of TNF and IL-1β, the effect of hBD2 on stimulation ofPBMC with a synthetic ligand (Pam3CSK4 (TLR2-TLR1 ligand), InvivoGentlrt-pms) was tested. hBD2 was able to downregulate TNF afterstimulation with this ligand as well, indicating that neutralization ofLPS or LTA is not responsible for the observed effect (results notshown). Moreover, stimulation of dendritic cells with a cytokinecocktail containing TNF-α and IL-α together with hBD2 had downregulatingeffect on IL-1β and IL-8 and IL-6 compared to stimulation with acytokine cocktail alone. Obviously no effect on TNF could be analyzed,due to stimulation with TNF-α (results not shown).

TABLE 6 TNF production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS or LTA with and without hBD2, allsamples tested on the same donor, representative experiment out of 5donors. TNF, pg/ml hBD2 hBD2 hBD2 (SD) Control 100 μg/ml 10 μg/ml 1μg/ml Medium 7.3 2.9 2.6 4.2 (5.9) (5.1) (6.6) (10.7) LPS 1708.6 634.21076.4 944.8 0.6 ng/ml (428.3) (226.1)*** (278.0)*** (326.6)*** LPS2572.1 1733.9 1306.6 1526.9 20 ng/ml (581.1) (461.3)*** (375.0)***(444.2)*** LTA 1097.4 375.2 494.7 711.5 1.25 μg/ml (293.8) (114.2)***(158.1)*** (282.5)*** TNF measured by Cytometric Bead Array (CBA) on aFACSarray, ***p < 0.001 compared to respective control (bold), analyzedby 2-way ANOVA (N = app. 200 for each data set).

TABLE 7 IL-1β production from human perifieral blood mononuclear cells(PBMC) after treatment with LPS or LTA with and without hBD2, allsamples tested on the same donor, representative experiment out of 5donors. IL-1β, pg/ml hBD2 hBD2 hBD2 (SD) Control 100 μg/ml 10 μg/ml 1μg/ml Medium 4.2 5.3 3.8 4.1 (4.7) (7.1) (5.8) (51.0) LPS 1734.3 81101949.8 1436.2 0.6 ng/ml (347.0) (454.4)*** (396.4)*** (429.7)*** LPS2629.5 1502.1 2273.9 1889.3 20 ng/ml (533.7) (407.5)*** (486.5)***(504.8)*** LTA 748.5 538.3 935.3 986.7 1.25 μg/ml (172.4) (137.3)***(238.0)*** (738.7)*** IL-1β measured by Cytometric bead array (CBA) on aFACSarray, ***p < 0.001 analyzed by 2-way ANOVA (N = app. 200 for eachdata set).

TABLE 8 IL-10 production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS or LTA with and without hBD2, allsamples tested on the same donor, representative experiment out of 5donors. IL-10, pg/ml hBD2 hBD2 hBD2 (SD) Control 100 μg/ml 10 μg/ml 1μg/ml Medium 2.09 2.9 1.6 2.09 (8.65) (4.6) (4.1) (4.3) LPS 63.15 232.7325.7 97.2 0.6 ng/ml (302.5) (61.5)*** (88.2)*** (31.1)* LPS 70.4 383.3355.8 111.3 20 ng/ml (22.8) (133.6)*** (99.5)*** (38.8)** LTA 14.0 175.6136.6 39.9 1.25 μg/ml (226.1) (57.0)*** (44.7)*** (16.9) IL-10 measuredby Cytometric bead array (CBA) on a FACSarray, ***p < 0.001, **p < 0.01,*p < 0.5 analyzed by 2-way ANOVA (N = app. 200 for each data set).

TABLE 9 PBMC viability after 24 h of stimulation measured by a MTSassay. Viability, MTS (Abs 490 nm hBD2 hBD2 hBD2 (SD)) Control 100 μg/ml10 μg/ml 1 μg/ml Medium 1.4 1.2 1.5 1.3 (0.2) (0.05)^(a) (0.2)^(a) (0.2)LPS 1.6 1.6 2.0 1.5 0.6 ng/ml (0.02) (0.1)^(ab) (0.2)^(b) (0.2) LPS 1.51.9 1.8 1.6 20 ng/ml (0.1) (0.2)^(b) (0.3)^(ab) (0.3) Values having adifferent subscript letter in rows are significantly different tested by2-way ANOVA followed by Bonferroni post-test.

TABLE 10 PBMC viability measured by Alamar Blue, one representativeexperiment out of 5 from 5 different donors. Viability, Alamar hBD2 hBD2hBD2 Blue (RFU (SD)) Control 100 μg/ml 10 μg/ml 1 μg/ml Medium 40971303950053 3683369 4064143  (166631)   (34466)^(a)   (355296)^(a)  (104634)LPS 4279424 4831188 4664362 4230588 0.6 ng/ml  (336188)   (67646)^(b)  (147776)^(b)  (139745) LPS 4604671 4765256 4623818 4561739 20 ng/ml (125840)   (41383)^(b)   (56643)^(b)  (138852) LTA 4018914 46641854677870 4148294 1.25 μg/ml   (632833)¹   (154023)^(b,2)   (10199)^(b,2)  (182730)¹² Values having a different superscript letter in rows andvalues having a different superscript number in columns aresignificantly different tested by 2-way ANOVA followed by Bonferronipost-test.

TABLE 11 TNF-alfa secretion from PBMC after stimulation with hBD2, LTA,LPS or combinations hereof. TNF-α, ng/ml hBD2 hBD2 hBD2 (SD) Control 100μg/ml 10 μg/ml 1 μg/ml Medium nd Nd Nd Nd LPS 0.99 0.41 0.59 0.70 0.6ng/ml (0.27) (0.03)** (0.08)* (0.18) LPS 1.44 0.53 0.49 1.18 20 ng/ml(0.31) (0.01)** (0.05)** (0.42) LTA 0.90 0.21 0.27 0.85 1.25 μg/ml(0.32) (0.05)* (0.04)* (0.29) TNF-alfa measured by ELISA, nd: notdetectable, detection limit in assay 0.01 ng/ml, *p < 0.05 compared torespective control, **p < 0.01 compared to respective control

TABLE 12 IL-10 secretion from PBMC after stimulation with hBD2, LTA, LPSor combinations hereof, TNF-alfa measured by ELISA, nd: not detectable,detection limit in assay 0.03 ng/ml IL-10, ng/ml hBD2 hBD2 hBD2 (SD)Control 100 μg/ml 10 μg/ml 1 μg/ml Medium nd Nd Nd Nd LPS nd 0.14 0.04Nd 0.6 ng/ml (0.04) (0.0) LPS nd 0.46 0.34 Nd 20 ng/ml (0.04) (0.04) LTAnd nd nd Nd 1.25 μg/ml

TABLE 13 IL-1β secretion from PBMC after stimulation with hBD2, LTA, LPSor combinations hereof, TNF-alfa measured by ELISA, nd: not detectable,detection limit in assay 0.016 ng/ml, **p < 0.01 compared to respectivecontrol IL-1β, ng/ml hBD2 hBD2 hBD2 (SD) Control 100 μg/ml 10 μg/ml 1μg/ml Medium nd Nd Nd Nd LPS 0.318 0.275 0.268 0.237 0.6 ng/ml (0.087)(0.015) (0.039) (0.007) LPS 0.920 0.395 0.354 0.638 20 ng/ml (0.267)(0.033)** (0.013)** (0.159) LTA 0.291 0.281 0.193 0.224 1.25 μg/ml(0.092) (0.059) (0.019) (0.030)

TABLE 14 TNF production from human peripheral blood mononuclear cells(PBMC) after treatment with PGN, with and without hBD2; all samplestested on the same donor. TNF, pg/ml hBD2 hBD2 hBD2 (SD) Control 100μg/ml 10 μg/ml 1 μg/ml Medium 0.0 3.6 3.7 3.4 (4.0) (5.3) (6.2) (5.2)PGN 1099.1 274.9 362.0 809.9 40 μg/ml (251.6) (71.6)*** (97.7)***(246.7)*** TNF measured by Cytometric Bead Array (CBA) on a FACSarray,***p < 0.001 compared to respective control, analyzed by 2-way ANOVA (N= app. 200 for each data set).

TABLE 15 IL-10 production from human peripheral blood mononuclear cells(PBMC) after treatment with PGN, with and without hBD2; all samplestested on the same donor. IL-10, pg/ml hBD2 hBD2 hBD2 (SD) Control 100μg/ml 10 μg/ml 1 μg/ml Medium 0.0 3.0 3.6 3.0 (4.1) (9.6) (13.1) (4.8)PGN 381.3 1054.2 523.4 337.8 40 μg/ml (92.3) (179.3)*** (111.5)***(89.1) TNF measured by Cytometric Bead Array (CBA) on a FACSarray, ***p< 0.001 compared to respective control, analyzed by 2-way ANOVA (N =app. 200 for each data set).

TABLE 16 TNF production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS or LTA, with and without hBD2 or twodifferent controls for inhibition of TNF (Dexamethasone andIndomethacin); all samples tested on the same donor. TNF, ng/ml LPS LPSLTA (SD) Medium 0.6 ng/ml 20 ng/ml 1.25 μg/ml Control 0.0 1.43 2.84 6.72(0.0) (0.05) (0.07) (0.14) Dexamethason 0.0  0.038 1.69 1.75 35 ng/ml(0.0)  (0.004) (0.05) (0.05) Dexamethason 0.0 0.30 0.91 2.05 3.5 ng/ml(0.0) (0.01) (0.03) (0.06) Dexamethason 0.0 0.61 6.04 4.73 0.35 ng/ml(0.0) (0.02) (0.14) (0.10) Indomethacin 0.0 1.71 2.70 5.80 7.2 ug/ml(0.0) (0.07) (0.07) (0.13) Indomethacin 0.0 1.56 7.54 5.50 0.72 ug/ml(0.0) (0.04) (0.17) (0.13) hBD2 0.0  0.003  0.000 0.11 1000 μg/ml (0.0) (0.002)  (0.002) (0.01) hBD2 0.0  0.000  0.038 1.15 100 μg/ml (0.0) (0.002)  (0.003) (0.04) hBD2 0.0 0.20 0.35 2.33 10 μg/ml (0.0) (0.01)(0.01) (0.06) hBD2 0.0 0.17 6.24 3.90 1 μg/ml (0.0) (0.01) (0.14) (0.10)TNF measured by Cytometric Bead Array (CBA) on a FACSarray, valuesunderlined are significantly reduced compared to respective control(bold), analyzed by 2-way ANOVA (N = app. 200 for each data set).

TABLE 17 IL-10 production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS or LTA, with and without hBD2 or twodifferent controls for antiinflammatory effects (Dexamethasone andIndomethacin); all samples tested on the same donor. IL-10, pg/ml LPSLPS LTA (SD) Medium 0.6 ng/ml 20 ng/ml 1.25 μg/ml Control 0.0 53.9 123.4  170.1  (218.8) (3.1) (4.6) (5.5) Dexamethason 0.0 100.4  152.5 175.2  35 ng/ml (1.4) (3.8) (5.2) (6.6) Dexamethason 2.7 64.6  122.8 112.5  3.5 ng/ml (1.9) (3.3) (4.7) (3.9) Dexamethason 3.9 46.8  197.1 126.6  0.35 ng/ml (1.9) (2.8) (7.2) (4.7) Indomethacin 0.0 45.7  77.9 90.4  7.2 ug/ml (1.5) (2.5) (3.6) (4.9) Indomethacin 0.0 37.3  108.0 84.9  0.72 ug/ml (1.4) (19.6)  (4.4) (3.5) hBD2 0.0 30.8  50.5  465.2 1000 μg/ml (1.6) (2.6) (3.2) (16.3)  hBD2 0.0 173.5  885.2  766.0  100μg/ml (4.9) (5.7) (22.2)  (21.7)  hBD2 3.9 165.1  497.5  355.8  10 μg/ml(1.7) (5.6) (13.5)  (9.4) hBD2 0.0 42.7  207.0  142.1  1 μg/ml (1.9)(2.8) (6.9) (4.9) IL-10 measured by Cytometric Bead Array (CBA) on aFACSarray, values underlined are significantly increased compared torespective control (bold), analyzed by 2-way ANOVA (N = app. 200 foreach data set).

TABLE 18 IL-1β production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS or LTA, with and without hBD2 or twodifferent controls for antiinflammatory effects (Dexamethasone andIndomethacin); all samples tested on the same donor. IL-1β, ng/ml LPSLPS LTA (SD) Medium 0.6 ng/ml 20 ng/ml 1.25 μg/ml Control 0.00 3.96 6.5811.47  (0.06) (0.18) (0.23) (0.38) Dexamethason 0.00 1.00 2.32 3.98 35ng/ml (0.00) (0.03) (0.07) (0.14) Dexamethason 0.00 1.90 3.58 5.22 3.5ng/ml (0.00) (0.06) (0.12) (0.19) Dexamethason 0.01 2.9  5.56 7.91 0.35ng/ml (0.00) (0.09) (0.18) (0.28) Indomethacin 0.04 4.1  6.12 8.91 7.2ug/ml (0.00) (0.13) (0.23) (0.30) Indomethacin 0.01 3.1  6.46 7.53 0.72ug/ml (0.00) (0.18) (0.22) (0.31) hBD2 0.01 0.53 1.19 4.43 1000 μg/ml(0.00) (0.02) (0.08) (0.14) hBD2 0.00 0.38 1.67 9.12 100 μg/ml (0.00)(0.01) (0.05) (0.32) hBD2 0.06 1.13 3.58 11.0  10 μg/ml (0.00) (0.04)(0.12) (0.37) hBD2 0.01 1.83 4.91 8.87 1 μg/ml (0.00) (0.06) (0.19)(0.29) IL-1β measured by Cytometric Bead Array (CBA) on a FACSarray,values underlined are significantly reduced compared to respectivecontrol (bold), analyzed by 2-way ANOVA (N = app. 200 for each dataset).

TABLE 19 TNF production in supernatant from a human monocyte cell line(MUTZ-3) after treatment with LPS or LTA, with and without hBD2. TNF,pg/ml hBD2 hBD2 hBD2 (SD) Control 100 μg/ml 10 μg/ml 1 μg/ml Medium 0.000.00 2.60 2.21 (5.56) (5.47) (7.17) (7.88) LPS 6.38 3.93 3.93 6.61 1.5μg/ml (9.28) (6.63)* (6.93)* (9.17) LTA 5.28 2.64 3.76 1.75 1.5 μg/ml(9.75) (29.19)* (7.72) (6.96)** TNF measured by Cytometric Bead Array(CBA) on a FACSarray, *p < 0.05 compared to respective control, **p <0.01 compared to respective control, analyzed by 2-way ANOVA (N = app.200 for each data set).

TABLE 20 TNF production in supernatants from immature dendritic cellsstimulated with LPS or LTA (to generate mature DC), with and withouthBD2. TNF, pg/ml hBD2 hBD2 hBD2 (SD) Control 100 μg/ml 10 μg/ml 1 μg/mlMedium 0.00 0.00 1.89 4.64 (1.74) (1.83) (2.15) (10.26) LPS 23.73 7.6613.8 18.04 1.5 μg/ml (3.28) (2.51)*** (2.33)*** (2.89)*** LTA 3.78 5.222.76 0.00 1.5 μg/ml (2.26) (2.25) (2.27)* (1.98)*** TNF measured byCytometric Bead Array (CBA) on a FACSarray, *significantly reduced p <0.05 compared to respective control, ***significantly reduced p < 0.01compared to respective control, analyzed by 2-way ANOVA (N = app. 200for each data set).

Example 5

Anti-Inflammatory Activity of hBD1, hBD2, hBD3, and a hBD4 Variant

Example 5 was carried out essentially as described in Example 4. Thecompound rhBD2, as shown in the tables below, is recombinant hBD2, whichis identical to hBD2 as used in Example 4.

The compounds hBD1, hBD2, hBD3 and hBD4 variant, as shown in the tablesbelow, were prepared using chemical synthesis, and obtained from PeptideInstitute Inc.

The amino acid sequence of recombinant hBD2 (rhBD2) is identical to theamino acid sequence of hBD2 prepared by chemical synthesis.

The hBD4 variant shown in the tables below consists of amino acids 3-39of hBD4, and the amino acid sequence is shown as SEQ ID NO:5.

In each table, all samples were tested on the same donor. SD meansstandard deviation.

Results

TABLE 21 TNF production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS with and without human beta defensins,dexamethasone or Infliximab. LPS LPS Medium 20 ng/ml 0.6 ng/ml TNF % ofTNF TNF Test pg/ml con- pg/ml % of pg/ml % of compound (SD) trol (SD)control (SD) control Medium 1 100% 2164  100% 728 100% (non- (1) (632)(156) treated) rhBD2 0 — 167 8%  74 10% 40 μg/ml (0)   (17)***    (5)***rhBD2 0 — 260 12% 125 17% 10 μg/ml (0)   (29)***   (20)** rhBD2 1 — 91842% 196 27%  1 μg/ml (0)   (373)***   (104)** hBD1 0 — 999 46%  91 13%40 μg/ml (0)   (116)***   (8)** hBD1 0 — 1311  61% 203 28% 10 μg/ml (1)  (417)***   (20)** hBD1 1 — 1395  64% 474 65%  1 μg/ml (1)   (201)***(187) hBD2 0 —  52 2% 176 24% 40 μg/ml (0)   (71)***  (103)** hBD2 0 —132 6% 304 42% 10 μg/ml (0)   (179)***  (108)* hBD2 0 — 411 19% 242 33% 1 μg/ml (0)   (581)***  (30)* HBD-3 0 — 451 21% 528 73%  1 μg/ml (0)  (24)***  (98) hBD4 0 — 139 6% 211 29% variant (0)    (6)***   (22)**10 μg/ml hBD4 0 — 778 36% 468 64% variant (0)   (27)***  (59)  1 μg/mlDexa- 0 — 635 29%  47 6% methasone (0)   (163)***    (8)*** Infliximab 0—  0 0%  0 0% (0)    (0)***    (0)*** TNF measured by Cytometric BeadArray (CBA) on a FACSarray, *p < 0.05, **p < 0.01, ***p < 0.001 analyzedby 2-way ANOVA and compared to non-treated cells by Bonferroniposttests.

TABLE 22 IL-10 production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS with and without human beta defensins,dexamethasone or Infliximab. LPS LPS Medium 20 ng/ml 0.6 ng/ml IL-10 %of IL-10 IL-10 Test pg/ml con- pg/ml % of pg/ml % of compound (SD) trol(SD) control (SD) control Medium 0 100% 111 100% 66 100% (non- (0)  (3) (5) treated) rhBD2 0 — 281 252% 108  162% 40 μg/ml (0)    (9)***  (4)*rhBD2 0 — 243 218% 103  155% 10 μg/ml (0)   (38)***  (14)* rhBD2 0 — 126113% 72 108%  1 μg/ml (0)  (14)  (9) hBD1 0 — 113 102% 69 104% 40 μg/ml(0)  (5)  (4) hBD1 0 — 100 90% 76 114% 10 μg/ml (0)  (1) (13) hBD1 0 — 95 85% 71 108%  1 μg/ml (0)  (17)  (6) hBD2 0 — 323 290% 131  197% 40μg/ml (0)    (0)***   (13)*** hBD2 0 — 240 215% 86 130% 10 μg/ml (0)   (0)***  (6) hBD2 0 — 123 110% 53 80%  1 μg/ml (0)  (0)  (5) hBD3 0 —152 137% 71 107%  1 μg/ml (0)  (72)*  (2) hBD4 0 — 187 168% 92 139%variant (0)    (9)*** (17) 10 μg/ml hBD4 0 — 175 157% 90 136% variant(0)    (8)*** (14)  1 μg/ml Dexa- 0 —  75 67% 47 70% methasone (0)  (6)*  (3) Infliximab 0 —  63 56% 46 69% (0)   (7)**  (9) IL-10measured by Cytometric Bead Array (CBA) on a FACSarray, *p < 0.05, **p <0.01, ***p < 0.001 analyzed by 2-way ANOVA and compared to non-treatedcells by Bonferroni posttests.

TABLE 23 IL-1β production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS with and without human beta defensins,dexamethasone or Infliximab. LPS LPS Medium 20 ng/ml 0.6 ng/ml IL-1β %of IL-1β IL-1β Test pg/ml con- pg/ml % of pg/ml % of compound (SD) trol(SD) control (SD) control Medium 0 100% 2544  100% 741 100% (non- (0)(226)  (93) treated) rhBD2 0 — 395 16% 124 17% 40 μg/ml (0)   (25)***  (11)*** rhBD2 0 — 624 25% 214 29% 10 μg/ml (0)   (37)***    (7)***rhBD2 0 — 1480  58% 284 38%  1 μg/ml (0)   (154)***   (15)*** hBD1 0 —1599  63% 302 41% 40 μg/ml (0)   (14)***    (3)*** hBD1 0 — 1913  75%401 54% 10 μg/ml (0)   (53)***   (17)*** hBD1 0 — 2087  82% 512 69%  1μg/ml (0)   (157)***   (45)** hBD2 1 — 316 12% 159 21% 40 μg/ml (1)   (0)***    (2)*** hBD2 0 — 589 23% 238 32% 10 μg/ml (0)   (0)***  (124)*** hBD2 0 — 1569  62% 312 42%  1 μg/ml (0)    (0)***   (28)***hBD3 0 — 568 22% 331 45%  1 μg/ml (0)   (126)***   (23)*** hBD4 0 — 46318% 163 22% variant (0)   (40)***    (5)*** 10 μg/ml hBD4 0 — 1004  40%286 39% variant (0)   (24)***   (11)***  1 μg/ml Dexa- 0 — 1120  44% 10414% methasone (0)   (220)***    (8)*** Infliximab 0 — 2704  106% 636 86%(0)  (0)  (81) IL-1β measured by Cytometric Bead Array (CBA) on aFACSarray, *** p < 0.001 analyzed by 2-way ANOVA and compared tonon-treated cells by Bonferroni posttests.

The effects of hBD1, hBD2, hBD3 and an hBD4 variant were tested on humanPBMC treated with and without LPG (Tables 21, 22 and 23). Forcomparison, rhBD2 was included in each setup.

TNF was downregulated for all defensins. The reduction in IL-113secretion was comparable to TNF, although not as pronounced as TNF.Secretion of IL-10 was significantly and dose-dependently enhanced forhBD2 and the hBD4 variant.

hBD3 was also tested at 10 μg/ml and 40 μg/ml and the hBD4 variant wasalso tested at 40 μg/ml; however, since both molecules were toxic to thecells at the these concentrations, it was not possible to discriminatebetween toxic and anti-inflammatory effects.

As a positive control on downregulation of TNF, two anti-inflammatorycompounds, Dexamethasone and Infliximab, were included in the setup.

Conclusion

All the tested human beta defensins showed anti-inflammatory potential.

Example 6

Reduction of IL-23 from Human Monocyte-Derived Dendritic Cells and HumanPBMCs

Example 6 was carried out essentially as described in Example 4 forhuman PBMCs; however, the readout was IL-23 instead of TNF, IL-1β andIL-10. Moreover, the effect of rhBD2 on human monocyte-derived dendriticcells was also investigated.

Generation of Monocyte-Derived Dendritic Cells (DCs)

The DCs were prepared according to a modified protocol originallydescribed by Romani et al. Briefly, peripheral blood mononuclear cells(PBMCs) were purified from buffy coats of healthy donors bycentrifugation over a Ficoll-pague (GE-healthcare) gradient. Monocyteswere isolated from PBMC by positive selection of CD14+ cells by magneticbeads (Dynal, Invitrogen) according to the manufacturer's instructions.The CD14+ monocytes were cultured in 6-well plates in RPMI/2% Human ABSerum recombinant human recombinant granulocyte-macrophagecolony-stimulating factor (GM-CSF, 20 ng/ml) and IL-4 (20ng/ml)(PeproTech) for 6 days, replenishing the medium/cytokines after 2and 5 days. After 6 days of culture the immature DCs are re-culturedinto 96-well plates in a concentration of 1×10⁶ cells/ml and leftuntreated or treated with a cocktail and/or hBD2 fore further 24 h. hBD2was tested in four concentrations in quadruplicate. hBD2 was analyzedfor its ability to suppress hDC-maturation into a proinflammatoryphenotype using a proinflammatory cocktail that contained LPS (100ng/ml) and IFN-γ (20 ng/ml). Dexamethasone was added 20 h prior to thecocktail as positive control for a compound with proven clinicalanti-inflammatory activity. The incubation with hBD2 was done 4 h priorto addition of cocktail.

Cytokine ELISA

Cell culture supernatants were collected and stored at −80° C. Amountsof IL-23 was measured by standard sandwich ELISA using commerciallyavailable antibodies and standards according to the manufacturer'sprotocols (eBioscience).

MTT Assay

A MTT based cell growth determination kit was used as a measure of cellsurvival after 48 h in order to evaluate if any of the cells wereseverely affected by treatment with vehicles, cocktail or hBD2 and wasdone according to the manufacturer's protocols (Sigma).

Statistical Analyses

All experiments were performed at least twice, with representativeresults shown. The data presented are expressed as mean plus/minusstandard deviation (SEM). Statistical significance was determined by2-way ANOVA with the variables being treatment (hBD2, dexamethazone,etc.) and stimulation (LPS, LTA, peptidoglycan, etc.) followed byBonferroni post-test as reported in the table legends. Differences wereconsidered significant for p<0.05.

Results

TABLE 24 IL-23 (pg/ml) in supernatants of human CD14⁺ monocyte-deriveddendritic cells stimulated with either medium (unstimulated), or LPS andIFN-γ and treated with either medium (untreated), hBD2 or Dexamehtasone,average (SEM), N = 4, one representative donor out of three. LPS (100ng/ml) and IL-23pg/ml(SEM) Unstimulated IFN-γ (20 ng/ml) Untreated 3753569  (96)  (130) hBD2 nd 3833 1 μg/ml  (88) hBD2 451 3308 10 μg/ml(121)  (169)* hBD2 nd 3042 30 μg/ml    (46)*** hBD2 nd 2145 100 μg/ml  (202)*** Dexamethasone 424 1147 1 μM  (38)   (268)*** *p < 0.05, **p <0.01, ***p < 0.001 analyzed by 2-way ANOVA and compared to non-treatedcells by Bonferroni posttests. nd: not detected (below detection limit).

TABLE 25 IL-23 (pg/ml) in supernatants of human PBMC stimulated witheither medium (control), 0.6 ng/ml LPS, 20 ng/ml LPS or 5 μg/ml LTA andtreated hBD2, Dexamehtasone or Infliximab, average (SEM). IL-23 pg/mlLPS LPS LTA (SEM) Control 0.6 ng/ml 20 ng/ml 5 μg/ml Control 257 553 510762 (non-treated)  (7)  (6)  (5)  (20) hBD2 218 338 263 383 1 μg/ml  (5)  (10)**   (5)**   (20)** hBD2 211 462 295 438 10 μg/ml  (4)   (2)*  (1)**   (9)** hBD2 207 484 488 810 100 μg/ml  (4)  (7)  (8)  (30)Dexamethasone 222 202 192 223 3.5 ng/ml  (5)   (5)**   (1)**   (1)**Infliximab 227 356 373 349 1 μg/ml  (10)   (10)**   (2)**   (1)** *p <0.05, **p < 0.01, ***p < 0.001 analyzed by 1-way ANOVA and compared tonon-treated cells by Dunnett's Multiple Comparison posttest.

As shown in Table 24, hBD2 suppresses significantly and dose-dependentlyIL-23 secretion from human CD14⁺ monocyte-derived dendritic cells.

For human PBMC, IL-23 secretion was also significantly suppressed (Table25). On these cells there was an inverse dose-dependency, that was foundto be a bell-shaped dose-response inhibition curve when testing lowerdoses of hBD2 (data not shown).

This shows that hBD2 might have a suppressive effect in a chronicautoimmune condition by suppression of IL-23 secretion, as IL-23 is animportant part of the inflammatory response. Th17 are dependent on IL-23for their survival and expansion, add Th17 cells have been shown to bepathogenic in several autoimmune diseases, such as Crohn's disease,ulcerative colitis, psoriasis and multiple sclerosis.

Example 7

Reduction of TNF Secretion from PBMCs with Mouse Beta Defensin 3 (mBD3)

Example 7 was carried out essentially as described in Example 4 forhuman PBMCs. Mouse beta defensin 3 (mBD3) was prepared using the sameprotocol as was used for production of hBD2 in Example 1. The amino acidsequence of mBD3 is shown in SEQ ID NO:6. Mouse PBMCs were prepared asdescribed below.

Isolation and Stimulation of Mouse Peripheral Blood Mononuclear Cells(PBMC)

Mouse peripheral blood mononuclear cells were isolated from blood of tenNMRI mice. In short, heparinized blood was diluted 1/1 v/v with RPMI andsubjected to Ficoll density centrifugation within 2 h of drawing. Plasmawas collected from the top and discarded. Isolated PBMC were resuspendedin culture medium (RPMI 1640 (Gibco, 42401) w/1% penicillin andstreptomycin and 1% L-Glutamine) and seeded in 90-well culture plateswith 115.500 cells per well in a total of 200 μl. PBMC from the samedonor were stimulated with 100, 10 or 1 μg/ml of hBD2 or mBD3 (mousebeta defensin 3); either alone or together with 20 ng/ml LPS (E. coli,O111:B4, Sigma L4391). Dexamethasone was added at 3.5 ng/ml to cultureswith and without LPS stimulation. The supernatants were collected afterincubation at 37° C. for 24 hours, and stored at −80° C. until cytokinemeasurement.

Cytokine production in supernatants was measured by flow cytometry witha mouse inflammation cytometric bead array (CBA) according tomanufacturer's instructions (BD) on a FACSarray flow cytometer.

Viability was measured by Alamar Blue (Biosource DALL 1100) aftersupernatant were collected.

Results

TABLE 26 TNF production from human peripheral blood mononuclear cells(PBMC) after treatment with LPS with and without hBD2, all samplestested on the same donor, representative experiment out of two donors.TNF pg/ml LPS (SEM) Medium 20 ng/ml Medium 5 1353  (1) (140) mBD3 2 3841 μg/ml (0)   (11)*** mBD3 2  51 10 μg/ml (0)    (1)*** mBD3 39  166 100μg/ml (19)    (17)*** hBD2 3 633 1 μg/ml (0)   (110)*** hBD2 2 359 10μg/ml (0)   (10)*** hBD2 2 342 100 μg/ml (0)   (34)*** Dexamethasone 1460 3.5 ng/ml (0)   (29)*** Infliximab 0  1 1 μg/ml (0)    (0)*** TNFmeasured by Cytometric Bead Array (CBA) on a FACSarray, ***p < 0.001compared to respective control, analyzed by 2-way ANOVA (N = 2).

TABLE 27 TNF production from mouse peripheral blood mononuclear cells(PBMC) after treatment with LPS with and without mBD3, all samplestested on the same donor, representative experiment out of two donors.TNF pg/ml LPS (SEM) Medium 20 ng/ml Medium 578 2063   (3)  (77) mBD3 3471600  1 μg/ml  (32)   (47)*** mBD3 180 297 10 μg/ml  (0)    (9)*** mBD3182 195 100 μg/ml  (5)    (6)*** Dexamethasone  94 328 3.5 ng/ml  (3)   (8)*** Infliximab 530 2119  1 μg/ml  (4)  (31) TNF measured byCytometric Bead Array (CBA) on a FACSarray, ***p < 0.001 compared torespective control, analyzed by 2-way ANOVA (N = 2).

As shown in Table 26, mouse beta defensin 3 (mBD3) is downregulating thesecretion of TNF from human PBMCs to the same extend as hBD2 anddexamethason. mBD3 also downregulate the secretion of TNF from mousePBMC (Table 27).

Accordingly, in this setup, mBD3 exhibits excellent anti-inflammatoryactivity.

REFERENCES

-   Bonoiotto et al., Human β-Defensin 2 induces a Vigorous Cytokine    Response in Peripheral Blood Mononuclear Cells. Antimicrobial Agents    and Chemotherapy (2006), 50: 1433-1441.-   Bowdish et al., Immunomodulatory properties of defensins and    cathelicidins. Curr. Top. Microbiol. Immunol. (2006) 306: 27-66.-   Gersemann et al., Crohn's disease—defect in innate defence. World J.    Gastroenterol. (2008) 14: 5499-5503.-   Lehrer, Primate defensins. Nat. Rev. Microbiol. (2004) 2: 727-738.-   Swidsinski et al. Mucosal flora in inflammatory bowel disease.    Gastroenterology (2002) 122: 44-54.-   Niyonsaba et al., Antimicrobial peptides human β-defensins stimulate    epidermal keratinocyte migration, proliferation and production of    proinflammatory cytokines and chemokines. Journal of Investigative    Dermatology (2007), 127: 594-604.-   Rowland et al., Differential regulation by thalidomide and    dexamethasone of cytokine expression in human peripheral blood    mononuclear cells. Immunopharmacology (1998), 40: 11-20.-   Wang et al., Host-microbe interaction: mechanisms of defensin    deficiency in Crohn's disease. Expert. Rev. Anti. Infect.    Ther. (2007) 5: 1049-1057.-   Wehkamp et al., Reduced Paneth cell alpha-defensins in ileal Crohn's    disease. Proc. Natl. Acad. Sci. U.S.A. (2005) 102: 18129-18134.

1-17. (canceled)
 18. A method of treating an inflammatory bowel disease,the method comprising administering orally to a subject in need of suchtreatment an effective amount of a mammal beta defensin.
 19. The methodof claim 18, wherein the effective amount is effective to reduceTNF-alpha activity in the treated tissues.
 20. The method of claim 18,wherein the mammal beta defensin is administered at a daily dosage offrom about 0.001 mg/kg body weight to about 10 mg/kg body weight. 21.The method of claim 18, wherein the mammal beta defensin is administeredat a daily dosage of from about 0.01 mg/kg body weight to about 10 mg/kgbody weight.
 22. The method of claim 18, wherein the mammal betadefensin is a human beta defensin.
 23. The method of claim 18, whereinthe mama al beta defensin has at least 80% identity to the amino acidsequence of SEQ ID NO:1.
 24. The method of claim 18, wherein the mammalbeta defensin has at least 80% identity to the amino acid sequence ofSEQ ID NO:2.
 25. The method of claim 18, wherein the mammal betadefensin has at least 80% identity to the amino acid sequence of SEQ IDNO:3.
 26. The method of claim 18, wherein the mammal beta defensin hasat least 80% identity to the amino acid sequence of SEQ ID NO:4.
 27. Themethod of claim 18, wherein the human beta defensin is human betadefensin
 1. 28. The method of claim 18, wherein the human beta defensinis human beta defensin
 2. 29. The method of claim 18, wherein the humanbeta defensin is human beta defensin
 3. 30. The method of claim 18,wherein the human beta defensin is human beta defensin 4.